Aging, defined as a decrease in reproduction rate with age, is a fundamental characteristic of all living organisms down to bacteria. Yet we know little about the causal molecular mechanisms of aging within the in vivo context of a wild-type organism. One of the prominent markers of aging is protein aggregation, associated with cellular degeneracy in many age-related diseases, although its in vivo dynamics and effect are poorly understood. We followed the appearance and inheritance of spontaneous protein aggregation within lineages of Escherichia coli grown under nonstressed conditions using time-lapse microscopy and a fluorescently tagged chaperone (IbpA) involved in aggregate processing. The fluorescent marker is shown to faithfully identify in vivo the localization of aggregated proteins, revealing their accumulation upon cell division in cells with older poles. This accretion is associated with >30% of the loss of reproductive ability (aging) in these cells relative to the new-pole progeny, devoid of parental inclusion bodies, that exhibit rejuvenation. This suggests an asymmetric strategy whereby dividing cells segregate damage at the expense of aging individuals, resulting in the perpetuation of the population.ibpA ͉ inclusion bodies ͉ protein aggregation ͉ small heat-shock protein

The rules of nucleic acid base-pairing have been used to construct nanoscale architectures and organize biomolecules, but little has been done to apply this technology in vivo. We designed and assembled multidimensional RNA structures and used them as scaffolds for the spatial organization of bacterial metabolism. Engineered RNA modules were assembled into discrete, one-dimensional, and two-dimensional scaffolds with distinct protein-docking sites and used to control the spatial organization of a hydrogen-producing pathway. We increased hydrogen output as a function of scaffold architecture. Rationally designed RNA assemblies can thus be used to construct functional architectures in vivo.

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called 'extended synthesis-dependent strand annealing' (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become 'sticky ends' that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.

Deinococcus radiodurans' extreme resistance to ionizing radiation, desiccation, and DNA-damaging chemicals involves a robust DNA repair that reassembles its shattered genome. The repair process requires diploidy and commences with an extensive exonucleolytic erosion of DNA fragments. Liberated single-stranded overhangs prime strand elongation on overlapping fragments and the elongated complementary strands reestablish chromosomal contiguity by annealing. We explored the interdependence of the DNA recombination and replication processes in the reconstitution of the D. radiodurans genome disintegrated by ionizing radiation. The priming of extensive DNA repair synthesis involves RecA and RadA proteins. DNA polymerase III is essential for the initiation of repair synthesis, whereas efficient elongation requires DNA polymerases I and III. Inactivation of both polymerases leads to degradation of DNA fragments and rapid cell death. The present in vivo characterization of key recombination and replication processes dissects the mechanism of DNA repair in heavily irradiated D. radiodurans.

Highlights d A new methodology to detect and quantify most translation errors in proteomes d Most amino acid substitutions result from mis-pairing between codons and anti-codons d Proteins' error rates are reduced at conserved and highly expressed proteins d Translation speed is negatively correlated with error rates

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria. Using time-lapse microscopy and a microfluidic set-up, we have assessed in Escherichia coli the vertical transmission of the amyloidosis caused by the synthetic bacterial model prionoid RepA-WH1 at single cell resolution within their lineage context. We identify in vivo the coexistence of two strain-like types of amyloid aggregates within a genetically identical population and a controlled homogeneous environment. The amyloids are either toxic globular particles or single comet-shaped aggregates that split during cytokinesis and exhibit milder toxicity. Both segregate and propagate in sublineages, yet show interconversion. ClpB (Hsp104) chaperone, key for spreading of yeast prions, has no effect on the dynamics of the two RepA-WH1 aggregates. However, the propagation of the comet-like species is DnaK (Hsp70)-dependent. The bacterial RepA-WH1 prionoid thus provides key qualitative and quantitative clues on the biology of intracellular amyloid proteinopathies.